Breast cancer cells with the cell surface marker expression profile of CD44? or expressing higher levels of aldehyde dehydrogenase 1 are described as cancer stem cells (CSCs). Recent studies have identified additional markers that can further distinguish multiple subpopulations of CD44? cells that are resistant to radiation or chemotherapy. For example, CD44? cells retaining higher levels of the dye PKH26 after cell division or with lower proteosome activity are resistant to radiation. CD44? cells expressing mammosphere-enriched genes are resistant to anti-estrogen and chemotherapies. Despite this enormous progress, very few CSC markers are direct therapeutic targets. Therefore, further refining of CD44? cells based on additional markers is essential to characterize and target intrinsically drug-resistant or metastasis-prone cancer cells. Our studies have identified a subpopulation of CD44? cells enriched for the expression of anthrax toxin receptor 1 (ANTXR1). ANTXR1 was also elevated in primary breast epithelial cells with stem cell phenotype compared to luminal progenitor or mature cells. Only a subpopulation of breast cancer cells in primary breast cancer expresses ANTXR1. Activation of ANTXR1 through its natural ligand C5A increased Wnt activity as measured by phosphorylation of GSK3 and increased mammosphere formation. Based on these observations, we hypothesize that ANTXR1 is a functional marker of CSCs/drug-resistant cancer cells that controls self-renewal of CSCs through Wnt pathway. Two aims are proposed: 1) Disrupt ANTXR1 signaling in normal and cancerous breast epithelial cells and investigate stemness and sensitivity to chemotherapy. 2) Investigate whether ANTXR1-positive cells are enriched for Wnt activity in breast cancers and whether co- expression of ANTXR1 and Wnt pathway molecules in cancer cells is associated with disease parameters. For the first aim, we will use ANTXR1 antagonists such as mutant anthrax protective antigen with high affinity for ANTXR1 or soluble extracellular domain of ANTXR1 to investigate the effects on mammosphere forming ability, self-renewal and sensitivity to the chemotherapeutic drug docetaxel both in vitro and in vivo. In the second aim, we will determine ANTXR1, phospho-LRP6 (Wnt receptor) and nuclear 2-catenin expression by immunohistochemistry in primary breast cancers and correlate the expression with breast cancer subtypes, nodal status, grade, and the Oncotype DX recurrence score. Results of this study will have a significant impact in predicting response to conventional therapy as well as developing therapies against drug-resistant breast cancer. Prior studies on ANTXR1 in cancer were focused mainly on tumor-associated endothelial cells. This study will delineate its function in cancer cells in the context of CSC hypothesis as well as Wnt pathway modulation in cancer cells. Based on the results, future R01 type proposals will be developed, which will focus on small molecules that bind to ANTXR1:LRP6 and inhibit Wnt signaling in cancer cells. PUBLIC HEALTH RELEVANCE: This proposal will determine the role of anthrax toxin receptor 1 (ANTXR1) in breast cancer stem cells. ANTXR1 is expressed at a higher level in breast cancer stem cells compared to mature cells and may control the function of Wnt pathway in cancer stem cells. Inhibitors of ANTXR1, such as mutant protective antigen of anthrax or soluble ANTXR1 may inhibit ANTXR1 activity and sensitize cancer stem cells to chemotherapy.